Caffeine- and amino acid-effects upon try(plus) revertant yield in U.V.-irradiated hcr(plus) and hcr (minus) mutants of E. coli B-r

Summary The influence of supplementation of the post-irradiation plating medium with caffeine and/or amino acids, upon both U.V.-induced killing and try⁺ reversion yield, has been studied in hcr⁺ and hcr^- derivatives of E. coli B/r tryptophan auxotroph WWP-2. All experiments have been carried out with stationary phase cells grown in aerated nutrient broth.In the hcr^- strain, caffeine causes no enhancement of either killing or try⁺ revertant yield. There is mutational enhancement by supplementation with a low level of tryptophan, and an even greater effect when supplementation is with tryptophan plus an additional pool of other amino acids.In the hcr⁺ strain, tryptophan and/or a pool of other amino acids cause an enhancement of try⁺ yield, as in the hcr^- strain. Caffeine causes lethal enhancement on several different media. There is an apparently straightforward dose enhancement by caffeine, of lethality and try⁺ revertant frequency, on media containing no, or only, tryptophan. On media containing, however, both a low level of tryptophan and an additional pool of other amino acids, caffeine causes a preferential enhancement of try⁺ revertant frequency over and above pure dose enhancement.These results suggest that U.V. may cause two types of lesion. One type may lead only to mutation, and is repaired by both the caffeine-insensitive MFD, and the caffeine-sensitive hcr, dark repair systems. This first type of lesion is protected from repair by the presence of a pool of amino acids. The second type of lesion is hypothesised to be capable of causing either lethality or mutation, is repaired only by the hcr dark-repair system, and is not subject to protection by an amino acid pool.This work was initiated at the Radiological Research Laboratories, Columbia University, New York.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

The role of the HCR system in the repair of lethal lesions ofBacillus subtilis phages and their transfecting DNA damaged by radiation and alkylating agents

The role of the HCR system in the repair of prelethal lesions induced by UV-light, γ-rays and alkylating agents was studied in theBacillus subtilis SPP1 phage, its thermosensitive mutants (N3, N73 endts ₁) and corresponding infectious DNA. The survival of phages and their transfecting DNA after treatment with UV light is substantially higher inhcr ⁺ cells than inhcr cells, the differences being more striking in intact phages than in their transfecting DNA’s. Repair inhibitors reduce the survival inhcr ⁺ cells: caffeine lowers the survival of UV-irradiated phage SPP1 in exponentially growinghcr ⁺ cells but has no effect on its survival in competenthcr ⁺ cells; acriflavin and ethidium bromide decrease the survival of UV-irradiated SPP1 phage in both exponentially growing and competenthcr ⁺ cells to the level of survival observed inhcr cells; moreover, ethidium bromide lowers the number of infective centres inhcr ⁺ cells of UV-irradiated DNA of the SPP1 phage. Repair inhibitors do not lower the survival of UV-irradiated phages or their DNA inhcr cells. The repair mechanism under study repairs effectively also lesions induced by polyfunctional alkylating agents in transfecting DNA’s ofB. subtilis phages but is not functional with lesions induced by these agents in free phages and lesions caused in phages and their DNA by ethyl methanesulphonate or γ-rays.     

Enhanced UV Sensitivity of Thiobacillus ferrooxidans Resulting from Caffeine and Acriflavine Treatment of Irradiated Cells

This study was aimed at identifying the roles of caffeine and acriflavine, two repair inhibitors, on UV sensitivity of iron-oxidizing Thiobacillus ferrooxidans ATCC 13728. The UV-dose response survival curve was inflected in nature, suggesting the population heterogeneity of the isolate. Caffeine and acriflavine potentiated the UV-induced killing of the organism. With the increase in concentrations of these compounds, the extent of survival decreased. Similarly, the inhibitory effects of caffeine and acriflavine increased with the increase in dose of UV-irradiation. The cells irradiated with 10 s (equivalent to 5.6 × 10⁻⁵ J/m²/s) of UV-exposure tended to become resistant to the inhibitory effects of caffeine and acriflavine, as evidenced by the time course study of recovery. The cells appear to stage a dramatic recovery from UV damage in the presence of caffeine (3.0 mg/ml) and acriflavine (20 μg/ml) over a period of 25–30h and 35–40h respectively, when grown in the presence of energy sources. Received: 4 December 2000/Accepted: 10 January 2001     

The mutagenic effect of ethyl methanesulfonate on a non-acid-fast strain ofMycobacterium phlei

Ethyl methanesulfonate was used for the induction of three types of mutants in a non-acidfast strain ofMycobacterium phlei. A total of 20 auxotrophie mutants was isolated. The mutants were isolated mostly when using doses yielding higher survival of the cells of the basic suspension. The auxotrophic mutants isolated required mostly amino acids, two mutants required purines and three mutants required vitamins. By determining the frequency of spontaneous reversions, it was found that 9 auxotrophic mutants could be used for further genetic studies. These included the following phenotypes: isoleucine⁻, leucine⁻, lysine⁻, nicotinic acid⁻, pyridoxine⁻, and xanthine⁻. Seven scotochromogenic mutants were isolated after ethyl methanesulfonate treatment. One was ochre, the remaining six were orange. Six achromogenic mutants were detected. Spontaneous auxotrophic mutants, scotochromogenic and achromogenic mutants were not isolated. The treatment with 0.2m ethyl methanesulfonate resulted in an increase in the frequency of STM-resistant mutants, the maximum phenotypic expression taking place after 72 hours cultivation in a liquid medium without the drug. The frequency of induced STM-resistant mutants varied within the range of 8.6.10⁻⁵–1.0.10⁻⁴ as compared with the frequency of spontaneous mutants 5.8.10⁻⁶–8.8.10⁻⁶.     

Evidence for repair of ultraviolet-induced damage in Cystobacter species (Myxobacterales).

Cystobacter species strain CK 1 does not grow with more than 0.2 microgram/ml acriflavine. Spontaneous two-step mutants growing with 2 microgram acriflavine per ml have been selected. One mutant (strain CK3) was used to investigate the effect of repair inhibitors. Both strains exhibit pronounced shoulders in their UV dose curves of inactivation. Acriflavine (AF), coumarin (CU), and caffeine (CA) when incorporated in the post-irradiation plating medium decreased survival of irradiated cells. Post-treatment with 2 microgram acriflavine/ml abolished the shoulder of the curve. Caffeine (1600 microgram/ml) and coumarin (350 microgram/ml) reduced it only to about 40%. It is concluded that probably two repair mechanisms are present. Pre-treatment of the cells with 2 microgram acriflavine/ml for two hours before UV-irradiation resulted in a constant dose enhancement factor of 1.9. The protective effect is increased with the time of treatment with acriflavine. This may indicate that pyrimidine dimers are responsible for UV-inactivation.     

Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.     

Differential expression of thermophilic phosphatases in the wild type and auxotrophic mutant strains of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

In the wild type strain (stock no. 1227) of Thermoactinomyces vulgaris, as reported earlier [Sinha and Singh (1980) Biochem. J. 190, 457–460], all phosphatase isoenzymes (three alkaline — AlpI, AlpII and AlpIII, and one acidic — Acp) are present. However, the auxotrophic mutants, the strains 1286 (thi ⁻), 1279 (nic ⁻, ura ⁻) and 1278 (thi ⁻, ura ⁻) exhibited two alkaline phosphatase isoenzymes (AlpII and AlpIII), but AlpI was lacking. In the strain 1261 (nic ⁻, thi ⁻), only AlpIII was expressed, and AlpI and AlpII isoenzymes were missing. The results suggest that the strains, which require either thiamine (1286 and 1278) or nicotinamide (1279) for their growth, were AlpI ⁻ mutants; and the strain (1261), which requires both thiamine and nicotinamide for its growth, was AlpI ⁻/AlpII ⁻ double mutant. There was no direct correlation between uracil auxotrophy and the expression of phosphatases. The uniform expression of AlpIII and Acp in all the strains, irrespective of their nutrient requirements, suggest that these constitutive phosphatases are species-specific. The specific activities of the thermophilic acid and alkaline phosphatases were maximum in the wild type strain (1227) of T. vulgaris. The next in phosphatase activity was the strain 1279 (an AlpI ⁻ mutant), followed by their decrease, in order, in the strains 1286 and 1278 (which were also AlpI ⁻ mutants); while least activity of these enzymes was observed in the obligate thermophile strain 1261 (AlpI ⁻/AlpII ⁻ double mutant).     

Adaptive mutation inEscherichia coli strain FC40

Mutations can arise in static populations of cells that are subjected to nonlethal selective pressure, a phenomenon that has been called ‘adaptive mutation’. This phenomenon has been extensively studied in FC40, a strain ofEscherichia coli that cannot metabolize lactose (Lac⁻) but that reverts to lactose utilization (Lac⁺) when lactose is its sole energy and carbon source. The adaptive Lac⁺ mutations arise by two mutational processes: a recombination-dependent process that is highly active on the episome carrying the Lac⁻ allele, and an unknown process that affects the whole genome. Most of the Lac⁺ mutations are due to the first process, which also produces nonselected mutations on the F′ episome. However, about 10% of the Lac⁺ mutations arise in a subpopulation of cells that experience a period of transient hypermutation. Although minor contributors to any one type of mutation, the hypermutators account for nearly all cases of multiple mutations. The evolutionary implications of these results are: (i) DNA synthesis associated with recombination may be an important source of spontaneous mutation, particularly in cells that are not actively growing; (ii) the efficient mutational mechanism that occurs on the episome could result in the horizontal transfer of new alleles among species that carry and exchange conjugal plasmids; and (iii) a subpopulation of transient hypermutators could be a source of multiple mutations that would allow for rapid adaptive evolution under adverse conditions.     

Mutagenic effect of ultraviolet radiation on the non-acid-fast strain ofMycobacterium phlei

The mutability of the PN strain ofMycobacterium phlei was examined after induction of auxotrophic mutants and of STM and VM-resistant mutants, by UV irradiation. A total of 30 auxotrophic mutants were isolated, most of them amino acid-dependent five purine-dependent, and one uracil-dependent. To induce the mutants higher UV doses had to be used so that the survival of cells in the original suspension would not exceed a few per cent. For further genetic work use can be made of 8 auxotrophic mutants (PN try^?ura^?, PN arg^?ura^?, PN ileu^?val^?, PN ileu^?, PN leu^?, PN lys^?, PN lys^?-VM^r, PN val^?), these showing a low frequency of spontaneous reversions. No spontaneous auxotrophic mutants have been found. The frequency of STM and VM-resistant mutants is increased upon UV irradiation, a post-irradiation incubation in a liquid medium without the drug being essential for their phenotypic expression. The highest increase of the number of these mutants is attained after 48 h of post-irradiation incubation and it has been found that, within a certain experimental scatter, the same frequency increase is found on using a complete or a minimal liquid medium. The frequency of spontaneous STM-resistant mutants lies within 5.8×10^?6–8.8×10^?6, of those VM-resistant between 3.1×10^?5 and 4.1×10^?5. The highest frequency of induced STM-resistant mutants lies between 3.0×10^?5 and 9.3×10^?5 and of VM-resistant mutants between 1.1×10^?4 and 2.2×10^?4     

Repair inhibition of potential mutations of ultraviolet-irradiatedEscherichia coli by chloroquine and quinacrine

The frequency of ultraviolet(UV)-induced mutations drops rapidly whenEscherichia coli Hcr⁺ cells (strains WP-2 Hcr⁺; B/r) are incubated on phosphate-buffered agar (PBA), but is reduced only slightly if chloroquine or quinacrine are incorporated into the medium. The excision-deficient WP-2 Hcr^– strain shows little reduction in the number of mutants when incubated on PBA. During postirradiation incubation on PBA, cell viability was relatively unaffected by the presence of the chemicals in the PBA (25 g/ml quinacrine; 50 g/ml chloroquine). When cells were given optimal doses of photoreactivating light, no further decline in mutations was obtained during subsequent incubation on PBA. Approximately 64% of the mutants seen when cells are treated with UV-PBA-chloroquine and 90% seen with UV-PBA-quinacrine can be repaired if cells are incubated on PBA. When these chemicals were added to the PBA, both excision-proficient strains (WP-2 Hcr⁺; B/r) demonstrated a marked reduction in the repair of UV-induced mutations to streptomycin resistance. Our results indicate that these chemicals interfere with the excision of UV-induced pyrimidine dimers, a process that normally occurs during postirradiation incubation on PBA.     

Isolation and Partial Characterization of Het− Fix− Mutant Strain of the Diazotrophic Cyanobacterium Anabaena variabilis Showing Chromatic Adaptation

We propose a model to describe the changes taking place in biochemical processes/events to explain the development of heterocyst and nitrogenase in a diazotrophic cyanobacterium Anabaena variabilis. For this purpose, a mutant strain of A. variabilis lacking heterocyst differentiation and incapable of growth with dinitrogen as the sole source of nitrogen has been isolated after nitrosoguanidine (NTG) mutagenesis and selection by penicillin enrichment. The mutant strain (Het⁻ Fix⁻) thus isolated has morphological variation and was incapable of reducing acetylene under anaerobic conditions, indicating its mutational loss of the process of nitrogen fixation. The Het⁻ Fix⁻ mutant strain had reduced glutamine synthetase (transferase) activity compared with its wild-type counterpart, suggesting a link between nif gene expression and the expression of gln A, the structural gene of GS. The Het⁻ Fix⁻ mutant strain compared with its wild-type strain also had an extremely high level of phycobiliprotein and a low level of carotenoids. Furthermore, the coiling of vegetative filaments in the Het⁻ Fix⁻ mutant strain, which reduced the surface area to be exposed to light, was a direct indication of the chromatic adaptation, because the mutant strain was found to be photosensitive, showing bleaching of the cells under high light intensity. Received: 13 December 2000 / Accepted: 9 February 2001     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Improvement of the multiple-stress tolerance of an ethanologenic <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by freeze-thaw treatment

An effective, simple, and convenient method to improve yeast’s multiple-stress tolerance, and ethanol production was developed. After an ethanologenic Saccharomyces cerevisiae strain SC521 was treated by nine cycles of freeze-thaw, a mutant FT9-11 strain with higher multiple-stress tolerance was isolated, whose viabilities under acetic acid, ethanol, freeze-thaw, H₂O₂, and heat-shock stresses were, respectively, 23-, 26-, 10- and 7-fold more than the parent strain at an initial value 2 × 10⁷ c.f.u. per ml. Ethanol production of FT9-11 was similar (91.5 g ethanol l⁻¹) to SC521 at 30°C with 200 g glucose l⁻¹, and was better than the parent strain at 37°C (72.5 g ethanol l⁻¹), with 300 (111 g ethanol l⁻¹) or with 400 (85 g ethanol l⁻¹) g glucose l⁻¹.     

Relative susceptibilities of caffeine-sensitive and caffeine-resistant strains of Candida albicans to inactivation and mutation by ultraviolet radiation

Summary Stable variants having increased resistance to growth inhibition by caffeine were obtained from four different absolute, amino acid auxotrophs of Candida albicans. Differences in growth rates and expression of auxotrophy between the resistant (Caf^R) variants and their sensitive (Caf^S) progenitors suggest that caffeine resistance arises through suppressor mutations which affect the fidelity of messenger RNA translation.Both Caf^S and Caf^R strains of C. albicans are more susceptible to inactivation by ultraviolet radiation (uv) when grown at 37°C rather than 25°C following exposure. Post irradiation growth on caffeine potentiates ultraviolet inactivation of all Caf^S strains at both temperatures. Depending on its origin, a Caf^R strain (i) may show greater, lesser or the same intrinsic susceptibility to uv inactivation as its Caf^S parent at 25°C or at 37°C and (ii) may or may not be refractory to post-irradiation contact with caffeine. Caf^R variants independently isolated from a given auxotroph are alike in inactivational responses whereas those obtained from different auxotrophs are dissimilar. This implies that different suppressor mutations are unique in the way they affect expression of potentially lethal uv damage and that only one kind of suppressor is obtained by selection for caffeine resistance in a particular auxotroph.The histidine requiring Caf^R strain WB-2CR is much more resistant to uv inactivation that its Caf^S parent WB-2. Moreover, post-irradiation survival of WB-2CR is unaffected by caffeine. However, WB-2CR and WB-2 are equally susceptible to uv-induced reversion to prototrophy. In both strains, caffeine does not enhance uv-induced reversion at 25°C or 37°C and exhibits an antimutagenic activity at high uv dosage at 37°C.The findings reinforce previously reported indications that, in C. albicans, (i) caffeine-sensitive excision-repair of uv damaged DNA does not occur and (ii) caffeine potentiates uv cellular inactivation by disturbing post-irradiation synthesis of protein essential for recovery from non-genetic damage.     

Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k)

 A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO₃ ⁻ metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO₃ ⁻, NO₂ ⁻ or NH⁺ ₄ as N-source. The mutants isolated absorb NO₃ ⁻ with rates close to those measured for the wild type and they excrete NO₂ ⁻ into the medium. The ratio between NO₃ ⁻ uptake and NO₂ ⁻ excretion was 1:1. The sensitivity of NO₃ ⁻ uptake to NH⁺ ₄ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds. Received: 8 October 1999 / Accepted: 25 January 2000     

Evidence against the involvement of phytochrome in UVB-induced inhibition of stem growth in green tomato plants

The effects of UVB on the kinetics of stem elongation of wild type (WT) and photomorphogenic mutants of tomato were studied by using linear voltage transducers connected to a computer. Twenty-one or twenty-six-day-old plants, grown in 12 h white light (150 μmol m⁻² s⁻¹ PAR)/12 h dark cycles, were first transferred to 200 μmol m⁻² s⁻¹ monochromatic yellow light for 12 h, then irradiated with 0.1 or 4.5 μmol m⁻² s⁻¹ UVB for 12 h and finally kept in darkness for another 24 h. The measurements of the kinetics of stem elongation started after 4 h under yellow light. Significant differences in stem growth during the irradiation with yellow light, as well as during the dark period, were found between the genotypes. In darkness, the magnitude of stem growth followed the order: tri > AC = fri > MMau > hp1. Two factors determined the large differences of growth in darkness: 1) the different stem elongation rate (SER) and 2) the different duration of the growing phase among the genotypes. In darkness the stem growth of au and hp1 mutants lasted for about 18 h, whereas it continued for the whole experimental period (36 h) in the other genotypes. UVB irradiation substantially reduced elongation growth of all genotypes (4.5 μmol m⁻² s⁻¹ being more effective than 0.1 μmol m⁻² s⁻¹). Both fluence rates of UVB induced a detectable reduction of SER already after 15 min of irradiation. Red light inhibited, while far red light promoted stem growth of all the genotypes tested. fri (phyA null), tri (phyB1 null), hp1 (exhibiting exaggerated phytochrome responses) mutants and WT tomato showed similar levels of UVB–induced inhibition of growth, while the aurea mutant showed the largest growth inhibition during the 12 h of irradiation. These results indicate that phytochrome is not directly involved in UVB control of stem elongation. The results of dichromatic irradiations UVB + red or UVB + far red indicate the presence of distinct and additive action of UVB photoreceptor and of the phytochrome system in the photoregulation of stem growth. This revised version was published online in June 2006 with corrections to the Cover Date.